Synthesis of ATP during oxidative phosphorylation occurs on the F1Fo-ATP synthase enzyme and accounts of the bulk of ATP synthesis in living cells. ATP synthase is an extraordinary enzyme because it acts as a molecular motor the energy of the transmembrane proton gradient is coupled through subunit rotation to the synthesis of ATP in three asymmetric but interconverting catalytic sites. In reverse, ATP hydrolysis drives subunit rotation and proton pumping. The long-term goal of this research is to understand the mechanism of F1Fo-ATP synthase in as much molecular detail as possible. The E. coli enzyme will be used because of its many advantages, e.g. it is readily amenable to molecular biology/genetic manipulations, it may be rapidly obtained in high yield, and it may be reconstituted in liposomes with excellent ATP synthesis activity. Specific goals are (1) determination of catalytic sites occupancy and nucleotide binding parameters in F1Fo in presence of a proton gradient, during ATP synthesis; (2) identification of functional interactions between gamma/alpha and gamma/beta subunits, by mutagenesis of residues in gamma which face alpha and beta; (3) elucidation of protein movements generated at the catalytic alpha/beta subunit interface as ATP hydrolysis proceeds through formation and collapse of the transition state to the ADP ground- state; (4) characterization of ATP hydrolysis at low ATP concentrations, where 120 degrees Celsius stepping of the rotor is seen; and (5) genetic analysis of the stator stalk, starting from mutations in alpha and delta subunits shown previously in this laboratory to interrupt both F1 binding to Fo and energy coupling. ATP-driven pumps are very widely distributed in nature, and are involved in many disease states. Work to be done here will consequently have broad impact in biology and medicine.